CZ304512B6 - Hyaluronic acid derivative, process for its preparation, modification process and use thereof - Google Patents

Abstract

The present invention relates to the process for preparing and use of {alpha}, {beta}-unsaturated aldehyde of hyaluronan having a double bond in the positions 4 and 5 and an aldehydic group in the position 6 of the glucosamine part of the polysaccharide according to the structural formula X or a hydrated form thereof according to the structural formula Y. The preparation process is based on dehydration of hyaluronan having an aldehydic group in the position 6 of the glucosamine part of the polysaccharide. Two preparation methods have been described and namely dehydration in a solution or heating in solid state in the absence of solvents, bases or other additives. This derivative allows stabilization of conjugates of hyaluronan with amino compounds by means of a multiple bond from the aldehyde side, and therefore, it is possible to effectively immobilize practically any compound containing an amino group to such modified hyaluronan in physiological conditions. In case of using a diamine or compounds or polymers containing three or more amino groups, it is possible to prepare crosslinked hyaluronan derivatives. The described solution brings along a significant advantage not only in the field of carriers of biologically active substances, but also in tissue engineering where cross linking with biologically acceptable amino compounds in physiological conditions is very much demanded.

Description

Hyaluronic acid derivative, its preparation, its modification and use

Technical field

The invention relates to the preparation and use of a novel hyaluronic acid derivative having a double bond at positions 4 and 5 of the glucosamine moiety of the polysaccharide and an aldehyde group at position 6 of the glucosamine moiety of polysaccharide of formula X, or a hydrated form thereof. bonded at positions 4 and 5 of the glucosamine moiety of the polysaccharide of formula ¥.

wherein R can be hydrogen, any metal cation or an organic cation.

This unsaturated aldehyde derivative of hyaluronan is suitable for binding compounds containing an amino group under physiological conditions. When the bound compound contains two or more amino groups, crosslinked materials can be prepared.

BACKGROUND OF THE INVENTION

Hyaluronic acid is a glycosaminoglycan composed of two repeating units of β- (1,3) -D-glucuronic acid and β- (1,4- N -acetyl-D-glucosamine).

Scheme 1 Hyaluronic acid

It has a large molecular weight of 5.10 ⁴ to 5.10 ⁶ g.mof ¹ , which depends on the method of isolation and the starting material. This highly hydrophilic polysaccharide is water-soluble as a salt over the entire pH range. It forms part of connective tissues, skin, synovial fluid of joints, plays a significant role in a number of biological processes such as hydration, organization of proteoglycans, cell differentiation, proliferation and angiogenesis. Because this polymer is intrinsic to the body and therefore biodegradable, it becomes a suitable substrate for tissue engineering or as a carrier for biologically active substances.

Modification of hyaluronic acid to HA-aldehyde

HA-aldehyde is most often prepared by selective oxidation of native hyaluronan. Oxidation of polysaccharides is a process in which the oxidation degree of the polysaccharide functional groups is changed. When an aldehyde is formed, the oxidation state is formally increased by one degree. Often, carboxylic acids (two-degree oxidation) are also formed, which may be

By-product of oxidation to aldehyde. In the case of hyaluronic acid, several approaches are known for preparing aldehyde-linked hyaluronan (HA-aldehyde). These hyaluronan derivatives are one of the most widely used precursors for the preparation of bio materials from chemically modified hyaluronan. The main reason is that the aldehyde groups are stable under physiological conditions, but still sufficiently reactive for a rapid and efficient chemical reaction with, for example, amines.

The main methods of preparing HA-aldehydes are described in Scheme 2 below.

Scheme 2

By far the most common method of introducing an aldehyde group to hyaluronan is by oxidation with NaIO ₄ in water (Scheme 2, Structure 1) (Spiro Robert et al .: WO 99/01 143, Aeschlimann Daniel, Bulpitt Paul: WO 2007/01 49 441). This modification results in the opening of the carbohydrate cycle to form two aldehyde groups.

Another method is to oxidize the primary hydroxyl group at the 6-glucosamine position of the polysaccharide to an aldehyde (Scheme 2, Structure 2) using NaClO / TEMPO in water (Buffa R., Kettou S., Velebný V. et al. WO 2011/069 475 ) or using the Dess-Martin periodate in DMSO (Buffa R., Kettou S., Velebny V. et al. WO 2011/069 474). In contrast to structure 1, the aldehyde group at this position retains the rigidity of the polymer chain.

An interesting way of introducing an aldehyde group to hyaluronan is to link this group via a linker (Scheme 2, Structure 3). Various approaches are possible here, for example the introduction of a vicinal diol to the carboxyl group of hyaluronan via an amide and subsequent oxidation of the diol with NaIO ₄ which gives the aldehyde bound via a linker (Hilbom J. et al: WO 2010/138 074). This strategy may have the advantage that the aldehyde group is sterically more accessible for possible further modifications.

In another application (Aeschlimann Daniel and Bulpitt Paul: WO 200701 49 441), the possibility of preparing HA-aldehyde by reducing the carboxyl group of hyaluronan using 9-BBN (9-borabicyclo [3.3.1] nonane) reagent was mentioned. The result is a hyaluronan with an aldehyde group at position 6 of the glucuron moiety of the polysaccharide (Scheme 2, Structure 4).

-2GB 304512 B6

Condensation of HA-aldehyde with N-nucleophiles

The main application advantage of the condensation of HA-aldehydes with N-nucleophiles (amines) is that it can be performed under physiological conditions. In general, the following scheme 3 describes this reaction:

Scheme 3

H ₂ N - X

HA-CHO -► HA-CH = N-X

The hydrolytic stability of the resulting imine -CH = N- linkage is largely dependent on the nature of the group X. When X is an atom that spends a free electron pair, for example -CH ₂ -, the resulting hydrolytically highly unstable imine HA-CH = N-CH ₂ -. When X is an atom carrying a free electron pair, a hydrolytically more stable conjugate is formed (oxime HA-CH = NO-, hydrazone HA-3H = N-NH-, semicarbazone HA CH = N NH C () and the like) where the imine is the CH = N- bond is stabilized by conjugation to the free electron pair of the X atom. Many patents are known describing the binding of amines of the formula NH ₂ -X-, where X is nitrogen or oxygen, to hyaluronan oxidized to aldehyde, where final materials are formed at physiologically acceptable conditions so they are applicable to a wide range of biomedical applications. More recently, a patent (Bergman K., et al: WO 2009/108 100) is claimed where hyaluronic acid materials modified with electrophilic groups such as aldehyde, maleimide, acrylate, acrylamide, methacrylate, methacrylamide, vinyl sulfone and aziridine are generally claimed. Crosslinking nucleophiles include hydrazides, semicarbazides, thiosemicarbazides, aminooxy, thiol and β-aminothiol groups. Similar is another application of the invention (Hilbom J. et al: WO 2010/138 074) describing the binding of N, S or simultaneously N and S nucleophiles directly to aldehyde-oxidized hyaluronan by oxidation with sodium periodate.

When X is an aliphatic carbon (Scheme 3), it is generally known that the imines formed are not hydrolytically stable (the -C = N- bond has no conjugation partner) and reversibly to the starting aldehyde and the amine (Buffa R., Kettou S., Velebný V. et al (WO 2011/069 474). The situation is described in Figure 4.

Scheme 4

Another possibility to stabilize said imines is to extend the conjugation from the opposite side, i.e. from the aldehyde side, i.e. to give the resulting imine conjugation with a multiple -C = C- bond. The general reaction is described in Scheme 5.

Scheme 5

HA-C = C-CHO

H ₂ N - X

-►

HA-C = C-CH = N-X

-3GB 304512 B6

This approach is rarely mentioned in the literature, for example for the reaction of aromatic aldehydes with amines to form the so-called Schiff bases, where stability is supported by conjugation with the aromatic cycle Ar-CHO + H ₂ NR -> Ar-CH = NR. In the case of polysaccharides or polymers in general, however, no analogous example has been found. With this modification of the polymers, it would be necessary to introduce an aromatic group on the aldehyde or generally some conjugated multiple linkages via a linker, which is a technological complication and the biocompatibility of the material is not guaranteed. However, this method opens up another potential complication. In the presence of an aromatic system or multiple conjugated multiple bonds, the material can already absorb in the visible region so that the substance will be colored, which is generally not desirable (possibly photosensitivity, complications in in vitro analytics studies).

SUMMARY OF THE INVENTION

The present invention provides hyaluronic acid of structural formula X or Y having modified some glucosamine cycles of the double bonded polysaccharides at positions 4 and 5, and at the same time an aldehyde group or geminal diol (structure Y) at position 6 of the glucosamine moiety of the polysaccharides is present

wherein R can be hydrogen, any metal cation or an organic cation. A non-limiting example of R is, for example, a sodium, potassium, calcium, or tetrabutylammonium or organic protonated triethylamine cation. The derivative preferably has a molecular weight in the range of 1x10 ³ to 5x10 ⁵ g.moT ¹ .

This solution makes it possible to stabilize conjugates of hyaluronan with amino compounds by means of a multiple bond from the aldehyde side so that virtually any compound containing an amino group can be bound to the hyaluronan thus modified.

This is a significant difference from the saturated aldehydes of hyaluronan, which are capable of firmly binding under physiological conditions the compounds of formula H ₂ NX-, where X is an atom bearing a free electron pair, usually oxygen or nitrogen. Since few natural substances contain the H ₂ NX- grouping, the solution described in the present invention gives a great advantage not only as a potential carrier of biologically active substances but also in tissue engineering where hyaluronan derivatives crosslinked under physiological conditions with biologically acceptable amino compounds are very often used. .

Furthermore, the invention relates to a process for the preparation of a derivative of structural formula X or Y, wherein first hyaluronic acid is oxidized to HA-aldehyde at position 6 of the glucosamine moiety (hereinafter referred to as Step 1) and then HA-aldehyde is dehydrated either in solution or by simple heating in the absence of solvents, bases or other additives (hereinafter referred to as Step 2). These two steps are discussed in more detail below:

Step 1:

Selective oxidation of the primary hydroxyl group of hyaluronic acid at position 6 of the glucosamine moiety of the polysaccharide to aldehyde. The reaction can be carried out, for example, using an oxidation system of the 2,2,6,6-tetramethyl-1-piperidinyloxy group of the R'-TEMPO / NaClO radical in water, wherein R ¹ is hydrogen or a V-acetyl group:

oxidation

H ₂ O

This step is preferably carried out in water at a temperature of -5 to 10 ° C, the molar amount of NaClO being in the range of 0.05 to 0.7 eq. and the molar amount of R'-TEMPO is in the range of 0.005 to 0.2 eq. relative to the dimer of hyaluronic acid. The starting hyaluronic acid may have a molecular weight in the range of 1 x ^{10 4} to ⁶ x ^{10 6} g.moT ¹ .

Step 2:

Option 1: Dehydration of HA-aldehyde in a polar aprotic solvent and water at 30 to 80 ° C, preferably at 50 to 60 ° C, or Option 2: Dry pure HA-aldehyde dry to 50 to 100 ° C , preferably at 70 to 80 ° C.

The first possibility is dehydration in an aqueous-organic medium where the organic solvent is miscible with water and the solvent / water volume ratio is in the range of 3/1 to 1/2. Preferably, bases with limited nucleophilic properties can be used in this step, for example, organic bases such as triethylamine or N-diisopropyl-N-ethylamine, or inorganic bases such as Ca (OH) ₂ . The amount of base in the reaction is 0.01-20 equivalents calculated per hyaluronan dimer, preferably 5 to 10 equivalents. The base can promote elimination by cleaving the proton at the alpha position of the aldehyde (position 5 of the cycle) and the resulting carbanion eliminates the hydroxy group at position 4 to form a multiple bond. As organic solvents it is possible to use aprotic water-miscible polar solvents, preferably DMSO or sulfolane. The reaction is preferably carried out for 12 to 150 hours.

A second, technologically very attractive way to carry out step 2 is to heat the starting saturated aldehyde in the dry state without the presence of any additives to a higher temperature, preferably to 70 to 80 ° C for 12 hours to 10 days, preferably 4 to 5 days.

The invention further relates to the use of unsaturated HA-aldehyde for amine coupling. More particularly, the invention relates to a method of modifying a hyaluronic acid derivative according to formula X or Y, wherein the derivative is reacted with an amine of the formula H ₂ NR ² , wherein R ² is alkyl, aromatic, heteroaromatic,

-5C 304512 B6 linear or branched chain C 1 -C ₃₀ , optionally containing N, S or O atoms. The amine may be, for example, an amino acid, peptide or polymer that contains a free amino group, and such a polymer may be, for example, deacetylated hyaluronic acid, amino-linked hyaluronic acid via a linker, or gelatin, or other biologically acceptable polymer. The amount of amine, amino acid, peptide or free amino groups is preferably polymerized in the range of 0.05 to 2 equivalents relative to the hyaluronan dimer.

No specific conditions are required to prepare these conjugates. The reaction may be carried out in water, phosphate buffer or water-organic solvent at a temperature in the range of 20 to 60 ° C for 10 minutes to 150 h. The organic solvent may be selected from the group consisting of water miscible alcohols, particularly isopropanol or ethanol and water miscible polar aprotic solvents, in particular dimethylsulfoxide, the water content of the mixture being at least 50% by volume. The reaction runs smoothly under physiological conditions, for example in a phosphate buffer at pH = 7.4 and 37 ° C, with a wide range of different amines ranging from simple amino acids to complex peptides. It is also possible under these conditions to bind hydrazines, hydroxylamines, hydrazides, semicarbazides or thiosemicarbazides without problems. When compounds containing two or more amino groups are bound, it is possible to prepare insoluble crosslinked derivatives with a wide range of viscoelastic properties.

O

COONa //

ch ₃ co

The higher stability of the amine and unsaturated HA-aldehyde binding compared to its saturated analog allows to produce more stable and better crosslinked insoluble biomaterials based on hyaluronan. This statement is described in more detail in the Examples section of the invention, Example 21, where a saturated and unsaturated HA-aldehyde derivative is compared with a similar degree of substitution and molecular weight in terms of final theological properties after crosslinking with deacetylated hyaluronan.

Compared to the analogs mentioned in the "Background of the Invention", the proposed modification method is more advantageous in that it allows to bind more strongly to hyaluronic acid under physiological conditions a substantially wider range of amino-containing compounds. This is a great advantage for applications mainly in tissue engineering, where many biocompatible crosslinking amino-linkers can be used under physiological conditions and in the presence of living cells. The modified derivatives can be used, for example, for the preparation of crosslinked materials and hydrogels, for the preparation of tissue engineering materials or for biomedical applications. Polysaccharides or polymers containing amino groups can also be used for crosslinking. Also in the field of carriers of biologically active substances, the present invention can be advantageously used. The proposed method makes it possible to immobilize a wider range of biologically active amines (eg peptides) to hyaluronan, which can then be naturally released in native (active) form. It has been found that at lower pH the amine unsaturated HA-aldehyde bond is less hydrolytically stable, so that the conjugates prepared can also be used as pH-responsive biomaterials (carriers, gels ...). It has been shown that unsaturated HAaldehyde alone is not cytotoxic, so that its conjugates are a suitable candidate for various biomedical applications. Although one of skill in the art could have expected that conjugation by the aldehyde with a C = C-fold bond would lead to increased toxicity, because for example acrolein CH ₂ = CH-CHO is a highly toxic and irritant, it is not. The derivative of the invention has a double bond directly

-630304512 B6 in the polymer backbone (without linker) and the resulting substrate did not show toxic properties. The derivatives of the formula X or Y can be used for the preparation of anticancer materials, as carriers of biologically active substances in cosmetics and pharmaceuticals, or as carriers of biologically active substances with controlled release by pH change.

The implementation of the solution described in this invention is not technologically complicated and does not require the use of expensive chemicals, solvents or isolation procedures.

Overview of the figure in the drawing

Figure 1 shows an elastic material prepared according to Example 20.

DETAILED DESCRIPTION OF THE INVENTION

DS = degree of substitution = 100% * (molar amount of attached substituent or modified dimer) / (molar amount of all polysaccharide dimers)

As used herein, the equivalent (eq) refers to a hyaluronic acid dimer, unless otherwise indicated. Percentages are by weight unless otherwise indicated.

The molecular weight of the starting hyaluronic acid (source: CPN spol. Sr.o., Dolní Dobrouč, Czech Republic) is weight-average and was determined by SEC-MALLS method.

Example 1 Preparation of 6-glucosamine oxidized HA-aldehyde.

Oxidation of hyaluronic acid

To a 1% aqueous solution of hyaluronan (1 g, 2x10 ⁵ g.mol ¹ ) containing 1% NaCl, KBr 1%), TEMPO (0.01 eq) and NaHCO ₃ (20 eq.) Was successively added aqueous NaClO solution. (0.5 eq) under nitrogen. The mixture was stirred at -5 ° C for 12 h, then 0.1 g of ethanol was added and stirred for 1 h. The resulting solution was then diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCO ₃ ) 3 times 5 liters (once a day) and against distilled water 7 times 5 liters ( 2 times a day). The resulting solution was then evaporated and analyzed.

DS 10% (determined by NMR) 1 H NMR (D ₂ O) δ 5.26 (s, 1H, polymer-CH (OH) ₂ )

HSQC (D ₂ O) cross signal 5.26 ppm (1 H) - 90ppm ( ¹³ C) (polymer-C7 / (OH) ₂ )

Example 2 Dehydration of HA-aldehyde

To a 3% solution of HA-aldehyde (0.1 g, DS oxidation degree = 10%, Example 1) in water was added 6.7 mL of DMSO and DIPEA base (5 eq). The mixture was stirred at 40 ° C for 72 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 6% (determined by NMR), Mw = 11x10 ⁴ g.mol ^-1 (determined by SEC MALLS)

Ή NMR (D ₂ O) δ 9.24 (s, 1H, -CH = O), 6.32 (m, 1H, -C (C = CH-O))

UV-Vis (D ₂ O) 252 nm, π- π * α, β-unsaturated aldehyde transition

-7EN 304512 B6

Example 3 Dehydration of HA-aldehyde

To a 4% solution of HA-aldehyde (0.1 g, DS oxidation degree = 10%, Example 1) in water was added 7.5 mL of DMSO and DIPEA base (5 eq). The mixture was stirred at 50 ° C for 72 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 5% (determined from NMR, detailed Example 2)

Example 4 Dehydration of HA-aldehyde

To a 2% solution of HA-aldehyde (0.1 g, DS degree of oxidation = 10%, Example 1) in water was added 2.5 mL of DMSO and DIPEA base (5 eq). The mixture was stirred at 50 ° C for 72 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 2% (determined from NMR, Example 2 in detail)

Example 5 Dehydration of HA-aldehyde

To a 3% solution of HA-aldehyde (0.1 g, degree of oxidation DS = 10%, Example 1) in water was added 6.7 mL of sulfolane. The mixture was stirred at 60 ° C for 72 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 1% (determined from NMR, Example 2 in detail)

Example 6 Dehydration of HA-aldehyde

To a 3% solution of HA-aldehyde (0.1 g, degree of oxidation DS = 10%, Example 1) in water was added 6.7 mL of sulfolane and Et ₃ N base (5 eq). The mixture was stirred at 50 ° C for 72 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 5% (determined from NMR, detailed Example 2)

Example 7 Dehydration of HA-aldehyde

To a 3% solution of HA-aldehyde (0.1 g, degree of oxidation DS = 10%, Example 1) in water was added 6.7 mL of sulfolane and DIPEA base (2 eq). The mixture was stirred at 80 ° C for 12 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 2% (determined from NMR, Example 2 in detail)

Example 8 Dehydration of HA-aldehyde

To a 3% solution of HA-aldehyde (0.1 g, degree of oxidation DS = 10%, Example 1) in water was added 6.7 mL of sulfolane and Ca (OH) ₂ base (1 eq). The mixture was stirred at 30 ° C for 150 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 2% (determined from NMR, Example 2 in detail)

Example 9 Dehydration of HA-aldehyde

HA-aldehyde (0.1 g, DS degree of oxidation = 10%, Example 1) was heated in the solid state for 5 days at 80 ° C. It was then analyzed by NMR.

DS 3% (determined from NMR, Example 2 in detail)

-8EN 304512 B6

EXAMPLE 10 Dehydration of HA-aldehyde

HA-aldehyde (0.1 g, DS degree of oxidation = 10%, Example 1) was heated at 10 ° C for 12 h. It was then analyzed by NMR.

DS 2% (determined from NMR, Example 2 in detail)

Example 11 Dehydration of HA-aldehyde

HA-aldehyde (0.1 g, DS degree of oxidation = 10%, Example 1) was heated in the solid state for 10 days at 50 ° C. It was then analyzed by NMR.

DS 2% (determined from NMR, Example 2 in detail)

Example 12 Binding of amines to α, β-unsaturated HA-aldehyde

To a 1% solution of unsaturated HA-aldehyde (0.1 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added n-butylamine (2 eq). The mixture was stirred at 37 ° C for 5 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 5% (determined by NMR) 1 H NMR (D ₂ O) δ 7.74 (s, 1H, -CH = N-Bu), 5.68 (m, 1H, -C (C) -CH = N-Bu)

HSQC (D ₂ O) cross signal 7.74 ppm (1 H) - 158 ppm ( ¹³ C) -CH = N-Bu cross signal 5.68 ppm (* H) - 112 ppm ( ¹³ C) -C7f = C -CH = N-Bu

Example 13 Binding of amines to α, β-unsaturated HA-aldehyde

To a one percent solution of unsaturated HA-aldehyde (0.1 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added n-butylamine (0.05 eq). The mixture was stirred at 20 ° C for 150 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 2% (determined from NMR, detailed in Example 12)

Example 14 Binding of amines to α, β-unsaturated HA-aldehyde

To a 1% solution of unsaturated HA-aldehyde (0.1 g, degree of substitution DS = 6%, Example 2) in water was added n-butylamine (0.3 eq). The mixture was stirred at 60 ° C for 10 min. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 5% (determined from NMR, Example 12 in detail)

Example 15 Binding of lysine to α, β-unsaturated HA-aldehyde

To a one percent solution of unsaturated HA-aldehyde (0.1 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added lysine (0.3 eq). The mixture was stirred at 20 ° C for 24 h. The resulting solution was then precipitated with isopropanol / hexane and the solids dried in vacuo.

DS 5% (determined from NMR)

Δ NMR (D ₂ O) δ 7.76 (s, 1H, -Ctf = N-lysine), 5.65 (m, 1H, -C 7 / = C-CH = N-lysine)

-9EN 304512 B6

Example 16 Binding of the pentapeptide pal-KTTKS (palmytoyl-Lys-Thr-Thr-Lys-Ser) to α, β-unsaturated HA-aldehyde

To a one percent solution of unsaturated HA-aldehyde (0.1 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added 5 mL of IPA followed by a solution of substituted pentapeptide pal-KTTKS ( 0.1 eq) in 5 ml isopropanol. The mixture was stirred at 20 ° C for 72 h. The resulting solution was evaporated to a third volume on a rotary evaporator and then precipitated with isopropanol / hexane and the solid dried in vacuo.

DS 1% (determined by NMR) 1 H NMR (D ₂ O) δ 7.75 (s, 1H, -Ctf = N-peptide), 5.66 (m, IH, -C // = C-CH = N -peptide)

Example 17 Crosslinking of α, β-unsaturated HA-aldehyde by lysine

To a 5% solution of unsaturated HA-aldehyde (0.1 g, DS substitution degree = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added a 1% solution of lysine in water (0.1 eq). The mixture was stirred at 20 ° C for 24 h. An increase in the viscosity of the resulting solution was observed.

Example 18 Crosslinking of α, β-unsaturated HA-aldehyde by dihydrazidadipate

To a 5% solution of unsaturated HA-aldehyde (0.015 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added a 1% solution of dihydrazidadipate in water (0.1 eq). The mixture was stirred at 20 ° C for 24 h. An increase in the viscosity of the resulting solution was observed.

Example 19 Preparation of deacetylated hyaluronan

To a 3% solution of hyaluronan (1g, 83x10 ⁴ g-mol ¹ ) in hydrazine hydrate containing 30 g of hydrazine sulfate was added 65 mL of sulfolane and the mixture was heated at 70 ° C for 48 hours. The resulting solution is diluted to 0.2% with distilled water and dialyzed against the mixture (0.1% NaCl, 0.1% NaHCCE) 3 times 5 liters (1x daily) and against distilled water 7 times 5 liters (2x daily) . The resulting solution was then evaporated and analyzed.

DS 32% (determined from NMR), Mw 37x10 ³ g.moT ¹ (Determined by SEC-MALLS) 1 H NMR (1% NaOD in D ₂ O) δ 2.75 (s, IH, -C7T-NH ₂ )

Example 20 Crosslinking of α, β-unsaturated HA-aldehyde by deacetylated hyaluronan

To a 3% solution of unsaturated HA-aldehyde (0.025 g, degree of substitution DS = 6%, Example 2) in 0.1 M aqueous phosphate buffer at pH 7.4 was added a 3% solution of deacetylated hyaluronan (0.015 g, Example 19) νΟ, ΙΜ aqueous phosphate buffer at pH 7.4 (0.1 eq). The mixture was stirred at 20 ° C for 24 h. A significant increase in the viscosity of the resulting solution was observed.

Example 21 Comparison of mechanical and viscoelastic properties of hydrogels based on cross-linked α, β-unsaturated HA-aldehyde and cross-linked saturated HA-aldehyde.

- crosslinking with deacetylated hyaluronan

-10GB 304512 B6

Material 1: Unsaturated HA-aldehyde (0.06 g, DS = 6%, Mw = 11 x ^{10 4} g.mol \ Example 2) 3% solution in PBS pH 7.4 + deacetylated hyaluronan (0.02 g, Example 19) 3% solution in PBS pH 7.4.

Material 2: Saturated HA-aldehyde (0.06 g, DS = 7%, Mw = 1x10 ⁵ g.mol ^-1 ) 3% solution in PBS pH 7.4 + deacetylated hyaluronan (0.02 g, Example 19) 3 % solution in PBS pH 7.4.

Hydrogen samples were prepared from the materials described above by mixing and thoroughly homogenizing both components (3% solution of unsaturated HA-aldehyde in PBS / 3% solution of saturated HA-aldehyde and 3% solution of deacetylated hyaluronan in PBS). The samples were always aged for 240 minutes at room temperature, after which a homogeneous transparent gel was formed. All samples were of the same size and were measured under constant laboratory conditions (temperature, pressure, humidity).

The mechanical properties of the samples were determined. Specifically, it was the Young's modulus of compression indicating the hardness / elasticity of the material, the toughness indicating the strength of the sample, and how much energy the material was able to absorb without causing permanent deformation. Furthermore, between the compressive strengths, indicating the maximum load the material is able to absorb without causing permanent deformation, and within the viscoelastic properties, the shear modulus and loss angle.

Material j Young's module i Strength limit 1 Toughness Module j Loss angle number compression elasticity (kPa) (kPa) j (J / m ³ ) elasticity) δ (°) in skidding! ! (Bye) l 0.844 I, 382.06 | 29690 160 2.36 2 0.482 j ............ 309.29 1 ....... 19488 “55” 10.3

The results obtained in this example demonstrate the advantageous use of unsaturated HA-aldehyde over saturated HA-aldehyde in terms of preparing stiffer and tougher (better cross-linked) tissue engineering materials.

Claims (15)

PATENT CLAIMS Hyaluronic acid derivative modified by a double bond at positions 4 and 5 of the glucosamine moiety of the polysaccharide and simultaneously oxidized to the aldehyde at position 6 of the glucosamine moiety of the polysaccharide, according to structural formula X, or its hydrated form according to structural formula Y) CH ₃ CO wherein R is hydrogen, any metal cation or organic cation selected from the group consisting of an organic cation of tetrabutylammonium type or protonated triethylamine. Hyaluronic acid derivative according to claim 1, characterized in that it has a molecular weight in the range of 1 to 5 x ^{10 5} g.mol ^-1 and R is a sodium, potassium, calcium or an organic cation of tetrabutylammonium or protonated triethylamine. Method for the preparation of a hyaluronic acid derivative as defined in claim 1 or 2, characterized in that the hyaluronic acid is oxidized at the 6-position of the glucosamine moiety in a first step preferably by means of the R'-TEMPO / NaClO system, wherein R ¹ is hydrogen or Nacetyl; in water at -5 to 10 ° C, the molar amount of NaClO is in the range of 0.05 to 0.7 equivalents and the molar amount of R ¹ -TEMPO is in the range of 0.005 to 0.2 equivalents relative to the hyaluronic acid dimer, whereupon it is oxidized In the second step, the hyaluronic acid dehydrates at positions 4 and 5 of the glucosamine moiety in the water / polar mixture in an aprotic solvent at a temperature of 30 to 80 ° C, preferably at a temperature of 50 to 60 ° C. The method of claim 3, wherein the composition further comprises a base in an amount of 0.01 to 20 equivalents, preferably 5 to 10 equivalents, relative to the dimer of hyaluronic acid, wherein the base is selected from the group consisting of organic bases, e.g. triethylamine or diisopropylethylamine, or inorganic bases such as Ca (OH) ₂ . The method of claim 3, wherein the aprotic solvent is water miscible and includes, for example, DMSO or sulfolane, and the solvent / water volume ratio is in the range of 3/1 to 1/2. Process according to any one of claims 3 to 5, characterized in that the reaction takes place for 12 to 150 hours. Process according to any one of claims 3 to 6, characterized in that the oxidized hyaluronic acid in the second step dehydrates in the solid phase without the use of solvents or other additives by heating to a temperature of 50 to 100 ° C, preferably 70 to 80 ° C for 12 hours. hours to 10 days, preferably 4-5 days. Process according to any one of claims 3 to 7, characterized in that the starting hyaluronic acid has a molecular weight in the range of 1 x 10 ⁴ g.mol ^-1 to 5 x 10 ⁶ g.mol ^-1 . A method of modifying a hyaluronic acid derivative as defined in claim 1 or 2, wherein the derivative is reacted with an amine of the formula H ₂ NR ² , wherein R ² is an alkyl, aromatic, heteroaromatic, linear or branched chain C 1 to C ₃₀ , optionally containing N, S or O atoms. 10. A method of modifying a derivative of hyaluronic acid according to claim 9, wherein the derivative is reacted with an amino acid or a peptide. 11. A method of modifying a hyaluronic acid derivative according to claim 9 wherein the derivative is reacted with a polymer containing a free amino group. -11 GB 304512 B6 The method of modifying a hyaluronic acid derivative according to claim 11, wherein the polymer is, for example, deacetylated hyaluronic acid, amino-linked hyaluronic acid via a linker, or gelatin, or another biologically acceptable polymer. The modification process of any one of claims 9 to 12, wherein the amount of amine, amino acid, peptide or free amino groups of the polymer is in the range of 0.05 to 2 equivalents relative to the hyaluronic acid dimer. - 12GB 304512 B6 A method of modification according to any one of claims 9 to 13, wherein the reaction with the amine, amino acid, peptide or polymer containing the free amino group is carried out in water, in a phosphate buffer or in a water-organic solvent system at a temperature of 20 to 60 ° C for 10 minutes to 150 hours. The modification method according to claim 14, characterized in that the organic solvent is selected from the group consisting of water-miscible alcohols, in particular isopropanol or ethanol, and water miscible polar aprotic solvents, in particular dimethylsulfoxide, wherein the water content of the mixture is at least 50% by volume. . Use of derivatives as defined in any one of claims 1 and 2 as carriers of biologically active substances in cosmetics and pharmaceuticals or as carriers of biologically active substances with controlled pH release. Use of derivatives prepared as defined in any one of claims 9 to 15 for the preparation of crosslinked materials and hydrogels, for the preparation of tissue engineering or biomedical applications.